DNA sequencing-based HLA typing detects a B-cell ALL blast-specific mutation in HLA-A(*)2402 resulting in loss of HLA allele expression.
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Alleles↗
Biomedical subjects
D C Sayer
Publications and source records attributed to D C Sayer.
A multicenter international evaluation of single-tube amplification protocols for sequencing-based typing of HLA-DRB1 and HLA-DRB3,4,5.
We have described previously a novel single-tube polymerase chain reaction (PCR) amplification (STAmp) protocol for the efficient sequencing-based typing (SBT) of human leukocyte antigen (HLA)-DRB1. The PCR amplification mix includes primers to each of seven allele group-sequence motifs. We have applied this principle to the simultaneous SBT of HLA-DRB3, -DRB4, and -DRB5 using locus specific primers. We report here a multicenter international evaluation of the STAmp protocols performed as a component of the 13th International Histocompatibility Workshop. Identical amplification primer mixes, sequencing primers, and DNA were sent to participating laboratories. The primer mixes contained the amplification primers and the PCR buffer. Each laboratory was requested to amplify the DNA with the primer mixes and perform SBT on the resulting PCR protocols, using their own protocols, and return the typing results for analysis. The reported results indicated that the expected sequence could be obtained with a variety of PCR amplification and sequencing platforms and protocols. There were difficulties but these seemed unrelated to STAmp reagents and suggest that optimal SBT results can be obtained if bi-directional sequencing is performed and software is used for sequence verification and editing. This indicates that SBT by STAmp can be applied in many laboratories for high-throughput HLA-DRB1 and HLA-DRB3,4,5 SBT.
Alleles↗
Assign 2.0: software for the analysis of Phred quality values for quality control of HLA sequencing-based typing.
As improvements to DNA sequencing technology have resulted in increasing the throughput of DNA sequencing, the bottleneck for high throughput DNA sequencing-based typing (SBT) has shifted to sequence analysis, genotyping and quality control (QC). Consistent high-quality DNA sequence is required in order to reduce manual verification and editing of sequence electropherograms. However, identifying systematic changes in quality is difficult to achieve without the aid of sophisticated sequence analysis programs dedicated to this purpose. We describe a computer software program called Assign 2.0, which integrates sequence QC analysis and genotyping in order to facilitate high-throughput SBT. Assign 2.0 performs an analysis of Phred quality values in order to produce quality scores for a sample and a sequencing run. This enables sample-to-sample and run-to-run QC monitoring and provides a mechanism for the comparison of sequence quality between various genes, various reagents and various protocols with the aim of improving the overall quality of DNA sequence data. This, in turn, will result in reducing sequence analysis as a bottleneck for high-throughput SBT.
Alleles↗
Quality assessment program for genotypic antiretroviral testing improves detection of drug resistance mutations.
Genotypic antiretroviral testing is now widely used for the management of patients who are undergoing antiretroviral therapy for human immunodeficiency virus infection. The assays are complex, and there is considerable potential for variation between laboratories. Informative and ongoing quality assessment programs (QAPs) which address all aspects of testing are required. The panel distribution of clinical material is a critical component of QAPs. We report on the results and data from a recent panel. Four cryopreserved plasma samples from treated donors were distributed to nine laboratories. Three laboratories performed testing by commercial assays, and six laboratories used in-house assays, with one laboratory reporting results from two in-house assays. There was complete concordance between results for 95.9% of the nucleotide sequence and 94.5% of the amino acid sequence. Despite this overall high level of concordance, the degree of concordance at drug resistance mutation (DRM) sites when DRMs were present was considerably less (38% of DRM sites). Consequently, only 3 of the 10 methods reported 100% of DRMs as present. This elevated discrepancy rate is almost certainly a result of variability in the identification of mixtures of nucleotides (mixtures) at any site within the sequence. In addition, laboratories differed in the number of codons in the reverse transcriptase gene that were sequenced and their ability to amplify all samples. This panel distribution demonstrated a requirement for laboratory participation in ongoing QAPs and the optimization of assays with standards that contain mixtures.
Anti-HIV Agents↗
A quality control program for crossmatching procedures for solid organ transplantation. The participating laboratories of the Australasian and South Asian Tissue Typing Association.
The National Kidney Matching Scheme (NKMS) allows matching and allocation of donor organs throughout Australia. Sera from potential recipients are distributed to each interstate tissue typing laboratory on a monthly basis for crossmatching in the event of a cadaver donor. Therefore, it is essential there is consensus for results between these laboratories in order for donated organs to be allocated appropriately. A quality control program conducted under the auspices of ASEATTA was undertaken for (1) panel reactive antibody characterization; (2) routine T lymphocyte crossmatching; and (3) characterization of antibody isotype by DTT treatment. These aims were achieved by distribution of (1) six sera for the determination of PRA activity; (2) 20 scrambled trays including replicate dilutions of a strongly positive lymphocytotoxic serum, high titer monoclonal antibody and negative sera and; (3) 20 trays containing sera with IgG and/or IgM antibodies. These were then evaluated by each laboratory on a panel of T cells. There was concordance between laboratories for PRA levels and antibody characterization. However, there was considerable variation in crossmatch sensitivity and reproducibility, several laboratories had carryover and others could not detect weak IgM antibodies. These results demonstrate the utility and need for ongoing crossmatch exchange programs, particularly for laboratories participating in organ exchange programs.
Dithiothreitol↗
Population frequencies and putative haplotypes of the killer cell immunoglobulin-like receptor sequences and evidence for recombination.
BACKGROUND: The killer cell immunoglobulin-like receptors (KIR) are a family of receptors expressed on natural killer (NK) cells and some T cells. Class I HLA molecules on target cells are the ligands for the KIR receptors. The number of KIR genes has been reported to vary between individuals, resulting in different KIR haplotypes. There is little published data on the frequency of each KIR gene and the linkage disequilibrium between the genes. Because there is evidence that NK cells may be involved in bone marrow transplant rejection, we have determined the KIR gene frequencies and possible haplotypic arrangements by linkage disequilibrium analysis in an Australian population. METHODS: Controls, patients with leukemia, and unrelated bone marrow donor-recipient pairs were typed for the presence of 11 KIR genes by polymerase chain reaction-sequence specific priming. RESULTS: Ninety percent of the population was found to have a sufficient number and variety of KIR genes to detect any mismatch of HLA-A, -B, and -C alleles on NK target cells. The 11 KIR genes could be divided into two groups based on linkage disequilibrium between pairs of genes. Evidence for a recombination within the KIR gene complex is presented. CONCLUSION: Typing for the presence of particular KIR genes may be indicated for bone marrow donor-recipient pairs for whom a class I HLA mismatch is unavoidable.
Bone Marrow Transplantation↗
HLA-B*27 typing by sequence specific amplification without DNA extraction.
HLA-B27 appears to play a direct role in the pathogenesis of ankylosing spondylitis and almost all patients with this disease have HLA-B27. Therefore, a diagnosis of ankylosing spondylitis can virtually be excluded in the absence of HLA-B27. Many techniques have been used for HLA-B*27 typing. Of these, molecular methods are the most sensitive and specific but require extracted DNA as the testing material. A technique where HLA-B*27 is amplified directly from whole blood using sequence specific primers has been developed. This technique uses small sample volumes, is not restricted by choice of anticoagulant or sample age up to at least six weeks, and can be applied to other clinical polymerase chain reaction based procedures.
Blood Specimen Collection↗
Structure and polymorphism of two stress-activated protein kinase genes centromeric of the MHC: SAPK2a and SAPK4.
As MHC genes are potent determinants of susceptibility to immunopathological diseases, the mapping of SAPK2a (CSBP) and SAPK4 to chromosome 6p 21.2-21.3 suggested that these genes may mediate the effects of the MHC on disease. Here we describe the genomic structure and localisation of both genes approximately 2.3Mb centromeric of HLA-DP. Examination of the complete coding region and selected intronic regions of SAPK2a and SAPK4 from 22 human EBV-transformed B-cell lines of different MHC haplotypes and racial background revealed complete sequence conservation. There were no notable differences in levels of expression of SAPK2a and SAPK4 mRNA in cell lines of different MHC haplotypes or racial origin. Examination of the SAPK2a and SAPK4 sequences from two chimpanzees revealed 3 nucleotide differences between human and chimpanzee in each gene resulting in only one amino acid change in SAPK4, and 6 nucleotide substitutions plus 2 deletions in 600bp of intronic sequence from SAPK4. This highlights the selective pressure placed on these genes to maintain their protein sequence, but does not favour a role in genetic regulation of disease or provide evidence of linkage disequilibrium with the MHC.
Amino Acid Sequence↗